The basic helix-loop-helix (bHLH) transcription factor genes Hand1 and Mash2 are essential for placental development in mice. Hand1 promotes differentiation of trophoblast giant cells, whereas Mash2 is required for the maintenance of giant cell precursors, and its overexpression prevents giant cell differentiation. We found that Hand1 expression and Mash2 expression overlap in the ectoplacental cone and spongiotrophoblast, layers of the placenta that contain the giant cell precursors, indicating that the antagonistic activities of Hand1 and Mash2 must be coordinated. MASH2 and HAND1 both heterodimerize with E factors, bHLH proteins that are the DNA-binding partners for most class B bHLH factors and which are also expressed in the ectoplacental cone and spongiotrophoblast. In vitro, HAND1 could antagonize MASH2 function by competing for E-factor binding. However, the Hand1 mutant phenotype cannot be solely explained by ectopic activity of MASH2, as the Hand1 mutant phenotype was not altered by further mutation of Mash2. Interestingly, expression of E-factor genes (ITF2 and ALF1) was down-regulated in the trophoblast lineage prior to giant cell differentiation. Therefore, suppression of MASH2 function, required to allow giant cell differentiation, may occur in vivo by loss of its E-factor partner due to loss of its expression and/or competition from HAND1. In giant cells, where E-factor expression was not detected, HAND1 presumably associates with a different bHLH partner. This may account for the distinct functions of HAND1 in giant cells and their precursors. We conclude that development of the trophoblast lineage is regulated by the interacting functions of HAND1, MASH2, and their cofactors.The placenta is critical for the intrauterine survival of mammalian embryos. In mice, mutations that severely disrupt placentation or establishment of the chorioallantoic circulation result in embryonic lethality by day 10.5 of gestation (E10.5). Defects in placentation also contribute to diseases of human pregnancy, including spontaneous abortion and preeclampsia (11). However, surprisingly little is known regarding the molecular events that regulate development of the trophoblast cell lineage, the epithelial component of the placenta. At the blastocyst stage, trophoblast cells in contact with the inner cell mass (polar trophectoderm) continue to proliferate and later contribute to the chorion and ectoplacental cone (24). In contrast, trophoblast cells distal to the inner cell mass (mural trophectoderm) terminally differentiate to form primary trophoblast giant cells. While mitotically arrested, these cells undergo continued rounds of DNA synthesis (endocycles), thereby acquiring their characteristic giant polyploid nuclei (54). Secondary giant cells subsequently arise due to differentiation of precursor cells present in the ectoplacental cone and, later in gestation, the spongiotrophoblast (17). Trophoblast giant cells participate in a number of processes critical to a successful pregnancy, including blastocyst implantatio...

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